EP2931652B1 - Internalisation spécifique de nanoparticules dans des cages de protéines - Google Patents

Internalisation spécifique de nanoparticules dans des cages de protéines Download PDF

Info

Publication number
EP2931652B1
EP2931652B1 EP13862131.3A EP13862131A EP2931652B1 EP 2931652 B1 EP2931652 B1 EP 2931652B1 EP 13862131 A EP13862131 A EP 13862131A EP 2931652 B1 EP2931652 B1 EP 2931652B1
Authority
EP
European Patent Office
Prior art keywords
protein
nanoparticles
cage
protein cage
sequences
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
EP13862131.3A
Other languages
German (de)
English (en)
Other versions
EP2931652A4 (fr
EP2931652A1 (fr
Inventor
David Paramelle
Nikodem Tomczak
Paul FREE
Sierin LIM
Tao Peng
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Agency for Science Technology and Research Singapore
Nanyang Technological University
Original Assignee
Agency for Science Technology and Research Singapore
Nanyang Technological University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Agency for Science Technology and Research Singapore, Nanyang Technological University filed Critical Agency for Science Technology and Research Singapore
Publication of EP2931652A1 publication Critical patent/EP2931652A1/fr
Publication of EP2931652A4 publication Critical patent/EP2931652A4/fr
Application granted granted Critical
Publication of EP2931652B1 publication Critical patent/EP2931652B1/fr
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01012—Dihydrolipoyllysine-residue acetyltransferase (2.3.1.12)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/91045—Acyltransferases (2.3)
    • G01N2333/91051—Acyltransferases other than aminoacyltransferases (general) (2.3.1)
    • G01N2333/91057—Acyltransferases other than aminoacyltransferases (general) (2.3.1) with definite EC number (2.3.1.-)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
    • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
    • Y10T428/2991—Coated
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
    • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
    • Y10T428/2991—Coated
    • Y10T428/2998—Coated including synthetic resin or polymer

Definitions

  • the present invention generally relates to methods for making hybrid assemblies of nanoparticles into protein cages by encapsulation.
  • Such bioinorganic hybrids are useful in many technical fields such as for instance bioimaging and biosensing.
  • Peng and Lim (2011) (Biomacromolecules 12(9) 3131-3138 ) described trimer-based design of pH responsive protein cage results in soluble disassembled structures. Key amino acids were identified at the intratrimer and intertrimer interfaces, and histidine residues were introduced to these key sites to probe for their influences on the E2 protein assembly.
  • this method can be utilized for encapsulation of the nanoparticles without disassembly of the cages and without any need for in-situ synthesis of the nanoparticles.
  • the nanoparticle is able to diffuse into the protein cage and bind to modified parts of the protein cage without disturbing the protein structure.
  • the protein cage is genetically modified at predefined locations with one or more sequences of aminoacids, e.g., histidines.
  • sequences of histidines allow for the insertion of nanoparticles, e.g., gold nanoparticles, functionalized by nitrilotriacetic acid (NTA) or tris-NTA (TNTA).
  • nanoparticles e.g., gold nanoparticles
  • NTA nitrilotriacetic acid
  • TNTA tris-NTA
  • Also disclosed herein is a protein cage genetically modified at predefined locations with one or more sequences of histidines has been provided.
  • such protein cages can be used to insert nanoparticles into them which are functionalized by one or more ligands selected from nitrilotriacetic acid, a multiple nitrilotriacetic acid and their derivatives.
  • nanoparticle coated with ligands which are able to bind to histidines of the genetically modified protein cages has been obtained according to the invention.
  • these presynthesized nanoparticles are able to be inserted into the protein cage according to the invention via holes without any need of protein cage disassembly.
  • hybrid assembly obtained by inserting a nanoparticle according to the invention into a protein cage according to the invention or a hybrid assembly made according to a method according to the invention has been provided together with its use.
  • such hybrids can be obtained even for nanoparticles which would otherwise not be able to be inserted into the protein cages and their uses have been made possible.
  • the term "about”, in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • the modification of the protein is preferably done by genetically engineering.
  • Such methods are widely known ( Dalmau, M. et al., Biotechnol. Bioeng. 2008, 101, 654-64 ; Ren, D. et al., Adv. Funct. Mater. 2012, 22, 3170-3180 ).
  • Functionalization of the nanoparticle can be done by suitable presynthesis (e.g. coating with suitable ligands) that enable the binding of the nanoparticle to the specific predefined locations of the protein cage.
  • the protein cage is genetically modified at predefined locations with one or more sequences of histidines.
  • sequence of histidine is a polyhistidine chain of two or more histidines.
  • affinity between histidine moieties and certain ligands can be used according to the invention to achieve the binding in the protein cage.
  • a method wherein the nanoparticles are coated with ligands preferably surface mono-, bi- and multivalent metal chelators, for example bivalent nickel or cobalt chelating ligands such as iminodiacetic acid and nitrilotriacetic acid, which are able to bind to the histidines can then be combined with such genetically modified protein cage.
  • ligands preferably surface mono-, bi- and multivalent metal chelators, for example bivalent nickel or cobalt chelating ligands such as iminodiacetic acid and nitrilotriacetic acid, which are able to bind to the histidines
  • a surface ligand is selected from nitrilotriacetic acid, a multiple nitrilotriacetic acid and their derivatives. Nitrilotriacetic (NTA) and tris-NTA (TNTA) are particularly preferred.
  • NTA, TNTA and their derivatives and their ligand functions and poly-histidine sequences affinity are known ( E. Hochuli et al. Nature Biotechnology 1988, 6, 1321-1325 ; Huang, Z. et al., Bioconjug. Chem. 2009, 20, 1667-72 ) .
  • nanoparticles can be used, including for instance also quantum dots (QD) and superparamagnetic iron oxide nanoparticles (SPIONs).
  • QD quantum dots
  • SPIONs superparamagnetic iron oxide nanoparticles
  • the nanoparticle is a metal, a metal oxide, an elemental or compound semiconductor, a polymer, an elemental cluster or a nanodiamond.
  • a compound semiconductor is a semiconductor compound composed of elements from two or more different groups of the periodic table.
  • a nanodiamond is a nanocrystalline form of diamond.
  • the nanoparticle is a gold or silver nanoparticle.
  • the protein cage is made from the protein dihydrolipoyl acetyltransferase, but is not limited to such protein cage.
  • the dihydrolipoyl acetyltransferase (E2) is an enzyme of the pyruvate dehydrogenase multi-enzyme complex from Geobacillus stearothermophilus.
  • Also disclosed herein is a protein cage genetically modified at predefined locations with one or more sequences of histidines has been provided.
  • Such protein cage can be used in the methods according to the invention.
  • a protein cage according to the disclosure which after assembly displays accessible histidine residues in the interior surface of the cage is preferred.
  • the display of accessible histidine means that a binding with corresponding ligands (e.g. NTA or TNTA) in the inside of the cage is especially strong.
  • a preferred protein is a dihydrolipoyl acetyltransferase (E2) mentioned in relation to the inventive methods.
  • nanoparticle coated with ligands which are able to bind to poly-histidine sequences has been prepared and is claimed herewith.
  • Typical nanoparticles can comprise a metal, a metal oxide, an elemental or compound semiconductor, a polymer, an elemental cluster or a nanodiamond, but there is no limitation to such nanoparticles. In general all nanoparticles of suitable size can be used according to the invention.
  • the nanoparticles have a preferred size of 1 to 50 nm. Most preferably they have a size of about 1 - 25 nm.
  • the nanoparticles have a size and shape allowing direct internalization inside the protein cage via holes.
  • hybrid assembly obtained by inserting a nanoparticle according to the invention into a protein cage according to the invention or a hybrid assembly made according to a method according to the invention has been prepared.
  • the hybrid assembly consists of a nanoparticle encapsulated in a protein cage.
  • the hybrid assemblies have a use in bioimaging, biosensing and signal multiplexing.
  • the protein/nanoparticles assemblies can be directly applied in bioimaging and generator (light generation), transducer (optical modulation), or act as intermediate (via e.g. electron transfer processes ( Soto C.M., Ratna B.R., Curr. Op. Biotechnol., 2010, 21, 426-438 ).
  • the use of monodisperse nanoparticles allows for efficient signal multiplexing.
  • the described materials can be employed in catalysis in aqueous dispersions ( Mori K., Yamashita H., PhysChemChemPhys, 2010, 12, 14420-14432 )biosensing where the protein shell provides biocompatibility and colloidal stability, and the encapsulated nanoparticle is a signal
  • the materials described herein may find also application in nanoelectronics as elements of nanoscale memory devices, e.g. floating nanodot gate memory ( Yamashita I., J. Mater. Chem., 2008, 18, 3813-3820 ).
  • a protein cage which is able to correctly assemble after the genetic modification.
  • a protein cage as described above which displays accessible histidine residues in the interior surface of the cage.
  • a presynthesized nanoparticle coated with surface ligands which are providing colloidal stability and are able to bind to the histidine sequences in the protein cage.
  • the nanoparticles may include Au, Ag and other metals, metal oxides, elemental and compound semiconductors, polymers, elemental clusters, nanodiamonds etc.
  • a nanoparticle as described above has a size and shape allowing direct internalization inside the protein cage via its holes.
  • a hybrid assembly made of the nanoparticle described above, and a protein cage described above.
  • Such hybrid assembly is obtained without the disassembly of the protein cages and without any in-situ chemical reaction during the assembly except supramolecular binding of the nanoparticle surface ligands to the genetically modified protein sequences inside the protein cages.
  • Non-limiting examples of the invention and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.
  • the protein is the enzyme dihydrolipoyl acetyltransferase (E2) of the pyruvate dehydrogenasemulti-enzyme complex from Geobacillus stearothermophilus .
  • the E2 protein subunits self-associate into a dodecahedron cage forming a hollow core that consists of 60 identical units.
  • the hollow core can potentially be utilized for molecular encapsulation. Its three-dimensional structure shows this assembly is approximately 24 nm in diameter with 12 openings of about 5 nm each. These openings were used for internalization of well-designed and functionalized nanoparticles.
  • E2-WT wild-type E2 protein
  • a loop structure located inside the cage was identified as a potential mutation sites ( Figure 2 ).
  • the residues at this loop structure were replaced with several combinations of histidine residues (LoopHistidine - LH - mutants, LH4, LH 6, and H3LH3).
  • E2-WT loop sequence WT: ...P 377 IVRDGEIVA 386 ... Mutations with polyHis sequences : LH5: ...P 377 IVHHHHHIVA 386 ... LH6: ...P 377 IVHHHHHHIVA 388 ...
  • Table 1 DLS results of all loop mutants (nm). Results are obtained using size distribution by volume. The slightly larger peak values of E2-LH6 compared to the E2-LH5 are potentially because of variation in the purities of the samples rather than size differences.
  • the functional peptide was inserted to the interior surface of E2 protein cage to replace the original RDGE peptide loop.
  • the incorporation of non-native peptide did not influence the self-assembly of the protein cage structure.
  • 600 ⁇ L of 2 mM H-CV 3 T-ol in water mQ, 300 ⁇ L of 2 mM HS-C11-EG4 in water mQ and 100 ⁇ L of 2 mM HS-C 11 -EG 3 -NTA are mixed in a 25 mL glass vial.
  • 50 ⁇ L of a 1% Tween 20 in water mQ are added along with 9 mL of citrate colloidal gold (5 nm) and the solution mixed for 10 minutes.
  • 1 mL of PBS buffer 10x (pH 7.4) is added and the solution is mixed overnight at room temperature.
  • the sample After centrifugation of the nanoparticles (16 Krcm, 1 hour), removal of the solvent and dispersion of the nanoparticles in 500 ⁇ L of PBS (pH 7.4), the sample is purified by size exclusion chromatography G25 in 200 mM NaCl aqueous solvent. The nanoparticles collected are concentrated by centrifugation (16 Krcm, 1 hour) and the solvent exchange with PBS (pH 7.4).
  • the NTA functions of the purified nanoparticles are activated with addition of a 2.8 M Ni(NO 3 ) 2 aqueous solution (Ni(II) final concentration of 250 mM) and the sample mixed for 2 hours.
  • the nanoparticles are then purified by size exclusion chromatography G25 in 200 mM NaCl aqueous solvent.
  • the nanoparticles collected are concentrated by centrifugation (16 Krcm, 1 hour) and the solvent exchange with phosphate buffer pH 8 (50 mM NaH 2 PO 4 , 0.5 M NaCl). Final concentration of nanoparticles is estimated at 0.4 ⁇ M by UV-visible spectrophotometry.
  • the images obtained by TEM show evidence of internalization of the nanoparticles inside mutated poly-histidine protein cages ( Figure 4 - A, B, C, D ).
  • the synthesis of metal nanoparticles may fit the range of 1 - 15nm, with the E2 protein cage approximately 25 nm in size.
  • GE Healthcare Sepharose, Sephacryl, or Superdex size-exclusion chromatography media the separation of different sized nanomaterials may be possible.
  • Initial tests with Superdex 200 HR have allowed separating 5nm from 10nm nanoparticles, and 10nm from ⁇ 15nm nanoparticles.
  • Agarose gel electrophoresis has also been used to separate the nanoparticle cages from protein fragments, and is a useful purification or analysis tool.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Physics & Mathematics (AREA)
  • Nanotechnology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Condensed Matter Physics & Semiconductors (AREA)
  • General Physics & Mathematics (AREA)
  • Manufacturing & Machinery (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Claims (8)

  1. Procédé pour l'internalisation de nanoparticules dans des cages protéiques, comprenant les étapes suivantes :
    (i) modification génétique d'une cage protéique en des emplacements prédéfinis avec une ou plusieurs séquences d'histidines, de façon que des résidus d'histidine accessibles soient présentés sur la surface intérieure de la cage protéique,
    (ii) fonctionnalisation de nanoparticules de façon qu'elles soient capables de se lier à la ou aux séquences d'histidines aux emplacements prédéfinis spécifiques de la cage protéique, et
    (iii) introduction des nanoparticules à travers des trous dans la cage protéique ;
    dans lequel la cage protéique est faite de la protéine dihydrolipoyle acétyltransférase.
  2. Procédé selon la revendication 1, dans lequel les nanoparticules sont revêtues de ligands qui sont capables de se lier aux séquences de polyhistidine.
  3. Procédé selon la revendication 2, dans lequel le ligand de surface est choisi parmi l'acide nitrilotriacétique, un acide nitrilotriacétique multiple, et leurs dérivés.
  4. Procédé selon l'une quelconque des revendications 1 à 3, dans lequel la nanoparticule est un métal, un oxyde métallique, un semi-conducteur élémentaire ou composite, un polymère, un module élémentaire ou un nanodiamant, ou une nanoparticule d'or ou d'argent.
  5. Cage protéique de dihydrolipoyle acétyltransférase génétiquement modifiée des emplacements prédéfinis avec une ou plusieurs séquences d'histidines, dans laquelle, après assemblage, la cage présente des résidus d'histidine accessibles dans la surface intérieure de la cage protéique.
  6. Assemblage hybride comprenant une nanoparticule revêtue de ligands qui sont liés à des séquences de polyhistidine à l'intérieur d'une cage protéique de dihydrolipoyle acétyltransférase génétiquement modifiée des emplacements prédéfinis avec une ou plusieurs séquences d'histidines de façon qu'après assemblage la cage présente des résidus d'histidine accessibles dans la surface intérieure de la cage protéique.
  7. Assemblage hybride selon la revendication 6, dans lequel la nanoparticule est une nanoparticule revêtue choisie parmi un métal, un oxyde métallique, un semi-conducteur élémentaire ou composite, un polymère, un module élémentaire ou un nanodiamant.
  8. Utilisation de l'assemblage hybride selon la revendication 6 ou la revendication 7 en bioimagerie, biodétection ou multiplexage de signal, dans la catalyse de solutions aqueuses, ou en tant que dispositifs de mémoire à l'échelle nanométrique.
EP13862131.3A 2012-12-12 2013-12-10 Internalisation spécifique de nanoparticules dans des cages de protéines Active EP2931652B1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SG2012091351 2012-12-12
PCT/SG2013/000521 WO2014092646A1 (fr) 2012-12-12 2013-12-10 Internalisation spécifique de nanoparticules dans des cages de protéines

Publications (3)

Publication Number Publication Date
EP2931652A1 EP2931652A1 (fr) 2015-10-21
EP2931652A4 EP2931652A4 (fr) 2016-05-25
EP2931652B1 true EP2931652B1 (fr) 2018-11-28

Family

ID=54538008

Family Applications (1)

Application Number Title Priority Date Filing Date
EP13862131.3A Active EP2931652B1 (fr) 2012-12-12 2013-12-10 Internalisation spécifique de nanoparticules dans des cages de protéines

Country Status (3)

Country Link
US (1) US10035990B2 (fr)
EP (1) EP2931652B1 (fr)
WO (1) WO2014092646A1 (fr)

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004001019A2 (fr) * 2002-02-01 2003-12-31 Montana State University Nouvelles nanoparticules et leur utilisation
US20090226525A1 (en) 2007-04-09 2009-09-10 Chimeros Inc. Self-assembling nanoparticle drug delivery system
US8343497B2 (en) * 2008-10-12 2013-01-01 The Brigham And Women's Hospital, Inc. Targeting of antigen presenting cells with immunonanotherapeutics

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MERCÈ DALMAU ET AL: "pH-Triggered Disassembly in a Caged Protein Complex", BIOMACROMOLECULES, vol. 10, no. 12, 14 December 2009 (2009-12-14), US, pages 3199 - 3206, XP055339131, ISSN: 1525-7797, DOI: 10.1021/bm900674v *

Also Published As

Publication number Publication date
EP2931652A4 (fr) 2016-05-25
US20150329836A1 (en) 2015-11-19
US10035990B2 (en) 2018-07-31
WO2014092646A1 (fr) 2014-06-19
EP2931652A1 (fr) 2015-10-21

Similar Documents

Publication Publication Date Title
Su et al. Nanoporous core@ shell particles: Design, preparation, applications in bioadsorption and biocatalysis
Du et al. Disulfide‐bridged organosilica frameworks: designed, synthesis, redox‐triggered biodegradation, and nanobiomedical applications
Cai et al. Immuno-modified superparamagnetic nanoparticles via host–guest interactions for high-purity capture and mild release of exosomes
US7151047B2 (en) Stable, water-soluble quantum dot, method of preparation and conjugates thereof
CN1988892A (zh) 纳米粒子及其制备方法
CN105555856B (zh) 植物糖原纳米颗粒及其制造方法
US9067181B2 (en) Separation of nanoparticles
CN105188905B (zh) 物质包封微囊及其制备方法
Mansur et al. Biomolecule-quantum dot systems for bioconjugation applications
US20140356272A1 (en) Volume production method for uniformly sized silica nanoparticles
US11549941B2 (en) Nucleic acid-functionalized nanoparticles
JP2009107106A (ja) 不規則な表面構造の早期導入により高収率のバイオイメージングナノ粒子を製造する方法
WO2020090708A1 (fr) Procédé de production de ferritine contenant un composé organique renfermé dans celle-ci
JP2008540726A5 (fr)
Xu et al. PEG modification enhances the in vivo stability of bioactive proteins immobilized on magnetic nanoparticles
US9623381B2 (en) Separation of nanoparticles
Li et al. Gold nanocrystals: Optical properties, fine-tuning of the shape, and biomedical applications
EP2931652B1 (fr) Internalisation spécifique de nanoparticules dans des cages de protéines
Haroun et al. Functionalized multi-walled carbon nanotubes as emerging carrier for biological applications
US7834160B2 (en) Process for producing zinc oxide-protein complex
CN107881176A (zh) 标记外泌体的试剂及其制备方法和使用方法
US11186845B1 (en) Compositions comprising a nanoparticle, a molecular basket comprising cyclodextrin, and a chloroplast-targeting peptide and methods of use thereof
CN103665117B (zh) 一种高效纯化水溶性纳米银颗粒链霉亲和素偶联物的方法
CN109047791B (zh) 金纳米颗粒及其合成方法
JP5131680B2 (ja) 疎水化ポリ（γ−グルタミン酸）からなるナノ粒子の製造方法

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20150703

AK Designated contracting states

Kind code of ref document: A1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

AX Request for extension of the european patent

Extension state: BA ME

RIN1 Information on inventor provided before grant (corrected)

Inventor name: PENG, TAO

Inventor name: PARAMELLE, DAVID

Inventor name: FREE, PAUL

Inventor name: TOMCZAK, NIKODEM

Inventor name: LIM, SIERIN

DAX Request for extension of the european patent (deleted)
A4 Supplementary search report drawn up and despatched

Effective date: 20160426

RIC1 Information provided on ipc code assigned before grant

Ipc: B82Y 40/00 20110101AFI20160418BHEP

Ipc: C12N 9/10 20060101ALI20160418BHEP

17Q First examination report despatched

Effective date: 20170131

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

INTG Intention to grant announced

Effective date: 20180705

GRAS Grant fee paid

Free format text: ORIGINAL CODE: EPIDOSNIGR3

GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Kind code of ref document: B1

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

REG Reference to a national code

Ref country code: CH

Ref legal event code: EP

REG Reference to a national code

Ref country code: AT

Ref legal event code: REF

Ref document number: 1070008

Country of ref document: AT

Kind code of ref document: T

Effective date: 20181215

REG Reference to a national code

Ref country code: DE

Ref legal event code: R096

Ref document number: 602013047603

Country of ref document: DE

REG Reference to a national code

Ref country code: IE

Ref legal event code: FG4D

REG Reference to a national code

Ref country code: NL

Ref legal event code: MP

Effective date: 20181128

REG Reference to a national code

Ref country code: LT

Ref legal event code: MG4D

REG Reference to a national code

Ref country code: AT

Ref legal event code: MK05

Ref document number: 1070008

Country of ref document: AT

Kind code of ref document: T

Effective date: 20181128

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190228

Ref country code: LT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: HR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: IS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190328

Ref country code: LV

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: ES

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: BG

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190228

Ref country code: FI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: AT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190301

Ref country code: RS

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: AL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: SE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: PT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20190328

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: IT

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: DK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: CZ

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: PL

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

REG Reference to a national code

Ref country code: DE

Ref legal event code: R097

Ref document number: 602013047603

Country of ref document: DE

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MC

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: SK

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: LU

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20181210

Ref country code: RO

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: EE

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: SM

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

REG Reference to a national code

Ref country code: IE

Ref legal event code: MM4A

REG Reference to a national code

Ref country code: BE

Ref legal event code: MM

Effective date: 20181231

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: SI

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: IE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20181210

26N No opposition filed

Effective date: 20190829

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20181231

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CH

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20181231

Ref country code: LI

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20181231

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: MT

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20181210

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: TR

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CY

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT

Effective date: 20181128

Ref country code: MK

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 20181128

Ref country code: HU

Free format text: LAPSE BECAUSE OF FAILURE TO SUBMIT A TRANSLATION OF THE DESCRIPTION OR TO PAY THE FEE WITHIN THE PRESCRIBED TIME-LIMIT; INVALID AB INITIO

Effective date: 20131210

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: GB

Payment date: 20221118

Year of fee payment: 10

Ref country code: FR

Payment date: 20221216

Year of fee payment: 10

Ref country code: DE

Payment date: 20221121

Year of fee payment: 10

P01 Opt-out of the competence of the unified patent court (upc) registered

Effective date: 20230530